Antithrombotic compound

ABSTRACT

This application relates to a novel compound of formula (I) (or a pharmaceutically acceptable salt thereof) as defined herein, processes and Intermediates for its preparation, pharmaceutical formulations comprising the novel compound of formula (I), and the use of the compound of formula (I) as an antithrombotic agent.

This application is a 371 of PCT/US98/09311 filed May 7, 1998 whichclaims the benefit of U.S. Provisional Application No. 60/046,584, filedMay 15, 1997.

This invention relates to a heterocyclic ketone compound havingsurprisingly potent antithrombotic efficacy. Thus, the invention relatesto the new antithrombotic compound, pharmaceutical compositionscontaining the compound as an active ingredient, and the use of thecompound for prophylaxis or treatment of a thromboembolic disorder suchas venous thrombosis, pulmonary embolism, arterial thrombosis, inparticular myocardial ischemia, myocardial infarction or cerebralthrombosis, general hypercoagulable states and local hypercoagulablestates, such as following angioplasty and coronary bypass operations, orgeneralized tissue injury as it relates to the inflammatory process. Inaddition, the new compound is useful as an anticoagulant in in vitro andex vivo applications.

Certain peptidyl heterocycles are disclosed in U.S. Pat. No. 5,523,308as inhibitors of thrombin useful in the treatment of thrombin-relateddisorders. Surprisingly, the compound of the instant invention exhibitshigh potency in inhibition of both thrombin and factor Xa, as well ashighly potent antithrombotic efficacy.

According to the invention, there is provided a compound of formula I

Y—CO—X—CO—Arg—R  I

wherein

Arg is L-arginyl;

R is 2-benzothiazolyl;

X—CO— is L-prolyl or (S)-azetidin-2-carbonyl; and

Y—CO— is a group of formula IIa, IIb, IIc or IId;

or a pharmaceutically acceptable salt thereof.

A particular compound of formula I is one in which X—CO— is L-prolyl(Pro).

A more particular compound of formula I is one in which X—CO— is Pro andY—CO— is a group of formula IIa or IIb.

One preferred compound of formula I is one in which X—CO— is Pro andY—CO— is a group of formula IIa, which compound may be denoted as acompound of formula Ia.

The preferred species is described hereinbelow as Example 1, where it isisolated as the acid addition salt with sulfuric acid.

Another particular compound of formula I is one in which X—CO— is Proand Y—CO— is a group of formula IIb, which compound may be denoted as acompound of formula Ib.

In addition to a compound of formula I, the present invention provides apharmaceutical composition comprising a compound of formula I, or apharmaceutically acceptable salt thereof, in association with apharmaceutically acceptable carrier, diluent or excipient.

The present invention also provides a method of inhibiting thrombosis ina mammal comprising administering to a mammal in need of treatment, anantithrombotic dose of a compound of formula I.

As is further discussed below, a compound of the instant invention is apotent, direct inhibitor of one or both of the enzymes thrombin andfactor Xa of the coagulation cascade. Accordingly, the present inventionfurther provides a method of inhibiting thrombin comprisingadministering to a mammal in need of treatment, a thrombin inhibitingdose of a compound of formula I, as well as a method of inhibitingthrombin in an in vitro or ex vivo application. Similarly, the presentinvention further provides a method of inhibiting factor Xa comprisingadministering to a mammal in need thereof, a factor Xa inhibiting doseof a compound of formula I, as well as a method of inhibiting factor Xain an in vitro or ex vivo application.

It is preferred that the chiral center indicated by the asterisk in thegroup Y be of (R)-stereochemistry, corresponding to that of a D-aminoacid.

However, it is to be understood that the present invention encompasses acompound of formula I as a mixture of diastereomers, as well as in theform of an individual diastereomer, and that the present inventionencompasses a compound of formula I as a mixture of enantiomers, as wellas in the form of an individual enantiomer, any of which mixtures orform possesses antithrombotic properties, it being well known in the arthow to prepare or isolate particular forms and how to determineantithrombotic properties by standard tests including those describedbelow. Owing to the facile epimerization of the α-proton of the Argmoiety, adjacent to the benzoxazolyl keto group, it may be preferred touse the compound of formula I as a mixture of epimers at that center.

In addition, a compound of formula I (or a pharmaceutically acceptablesalt thereof) may form a solvate with water or an organic solvent.Further, the compound, salt or solvate thereof may exhibit polymorphism.The present invention also encompasses any such solvate, polymorphicform, or mixture thereof.

A compound of formula I may be made by processes which include processesknown in the chemical art for the production of structurally analogouscompounds or by a novel process described herein. Novel processes andintermediates for the manufacture of a compound of formula I as definedabove provide further feature of the invention and are illustrated bythe following procedures in which the meanings of the generic radicalsare as defined above, unless otherwise specified. It will be recognizedthat it may be preferred or necessary to prepare a compound of formula Iin which a functional group is protected using a conventional protectinggroup, then to remove the protecting group to provide the compound offormula I.

Thus, there is provided a process for preparing a compound of formula I(or a pharmaceutically acceptable salt thereof) as provided in any ofthe above descriptions which includes

oxidation of the alcohol of a corresponding compound of formula III

Y—CO—X—CO—Arg(OH)—R  III

in which Arg(OH) indicates that the carbonyl portion of the L-arginylgroup is replaced by a hydroxymethylene group;

whereafter, for any of the above procedures, when a functional group isprotected using a protecting group, removing the protecting group; and

whereafter, for any of the above procedures, when a pharmaceuticallyacceptable salt of a compound of formula I is required, it is obtainedby reacting the basic form of the compound of formula I with an acidaffording a physiologically acceptable counterion or by any otherconventional procedure, such as, for example, exchanging the counterionof a salt.

Conveniently, the oxidation is carried out in an inert solvent, usingoxalyl chloride, dimethyl sulfoxide and a tertiary amine, for example asdescribed below in Example 1. It generally is preferred that the aminogroups of Y—CO— and the Arg side chain be protected during theoxidation.

A compound corresponding to compound of formula I in which one or morefunctional groups is protected provides another aspect of the invention.Such a compound may be represented as a compound of Formula Ip

(PY)Y—CO—X—CO—Arg(p^(A))—R  Ip

which bears one or more of the protecting groups p^(A) and P^(Y) whereinp^(A) is an optional protecting group(s) for the guanidino moiety of theArg side chain and P^(Y) is an optional protecting group for the aminonitrogen of the perhydroisoquinoline moiety. A typical value for p^(A)is tosyl and for P^(Y) is benzyloxycarbonyl.

Conveniently, an alcohol of formula III is prepared in a protected formby coupling a protected form of an acid of formula IV

Y—CO—X—CO—OH  IV

with a protected form of the alcohol of formula V

H—Arg(OH)—R  V

using a conventional coupling method, for example the mixed anhydridemethod described in Example 1.

The (optionally protected) acid of formula IV may be made by aconventional method. For example, when Y—CO— is of formula IIa and X—CO—is prolyl, the protected acid of formula IV may be prepared as describedbelow in Example 1 or as described in EP 670310 at Example 85.Similarly, when Y—CO— is of formula IId or IIb and X—CO— is prolyl, theprotected acid of formula IV is disclosed at Example 80 or 82 of EP670310 or U.S. Pat. No. 5,436,229. The preparation of the amino alcoholof formula V in which the guanidino group bears an N-tosyl protectinggroup is described below in Example 1.

As mentioned above, the invention includes a pharmaceutically acceptablesalt of a compound of formula I, which possesses sufficiently basicfunctional groups to react with any of a number of inorganic and organicacids which afford a nontoxic anion to form a pharmaceuticallyacceptable salt. Acids commonly employed to form acid addition salts areinorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid, phosphoric acid, and the like, and organic acidssuch as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromobenzenesulfonic acid, succinic acid, citric acid, benzoic acid,acetic acid, and the like. Examples of such pharmaceutically acceptablesalts thus are the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite,phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycollate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like. Preferred pharmaceutically acceptable acidaddition salts include those formed with mineral acids such ashydrochloric acid, hydrobromic acid and sulfuric acid.

A compound of formula I is isolated best in the form of an acid additionsalt. A salt of the compound of formula I formed with an acid such asone of those mentioned above is useful as a pharmaceutically acceptablesalt for administration of the antithrombotic compound and forpreparation of a formulation of the compound. Other acid addition saltsmay be prepared and used in the isolation and purification of thecompound.

The compound of formula I is believed to selectively inhibit thrombinand factor Xa over other proteinases and nonenzyme proteins involved inblood coagulation without appreciable interference with the body'snatural clot lysing ability (the compounds have a low inhibitory effecton fibrinolysis). Further, such selectivity is believed to permit usewith thrombolytic agents without substantial interference withthrombolysis and fibrinolysis.

In another of its aspects, the invention provides a method of treating athromboembolic disorder comprising administering to a mammal in need oftreatment an effective (thromboembolic disorder therapeutic and/orprophylactic amount) dose of a compound of formula I.

The invention in another of its aspects provides a method of inhibitingcoagulation in mammals comprising administering to a mammal in need oftreatment an effective (coagulation inhibiting) dose of a compound offormula I.

The coagulation inhibition and thromboembolic disorder treatmentcontemplated by the present method includes both medical therapeuticand/or prophylactic treatment as appropriate.

In a further embodiment the invention relates to treatment, in a humanor animal, of conditions where inhibition of thrombin or factor Xa isrequired. The compound of the invention is expected to be useful inmammals, including man, in treatment or prophylaxis of thrombosis andhypercoagulability in blood and tissues. Disorders in which thecompounds have a potential utility are in treatment or prophylaxis ofthrombosis and hypercoagulability in blood and tissues. Disorders inwhich the compound has a potential utility, in treatment and/orprophylaxis, include venous thrombosis and pulmonary embolism, arterialthrombosis, such as in myocardial ischemia, myocardial infarction,unstable angina, thrombosis-based stroke and peripheral arterialthrombosis. Further, the compound has expected utility in the treatmentor prophylaxis of atherosclerotic disorders (diseases) such as coronaryarterial disease, cerebral arterial disease and peripheral arterialdisease. Further, the compound is expected to be useful together withthrombolytics in myocardial infarction. Further, the compound hasexpected utility in prophylaxis for reocclusion after thrombolysis,percutaneous transluminal angioplasty (PTCA) and coronary bypassoperations. Further, the compound has expected utility in prevention ofrethrombosis after microsurgery. Further, the compound is expected to beuseful in anticoagulant treatment in connection with artificial organsand cardiac valves. Further, the compound has expected utility inanticoagulant treatment in hemodialysis and disseminated intravascularcoagulation. A further expected utility is in rinsing of catheters andmechanical devices used in patients in vivo, and as an anticoagulant forpreservation of blood, plasma and other blood products in vitro. Stillfurther, the compound has expected utility in other diseases where bloodcoagulation could be a fundamental contributing process or a source ofsecondary pathology, such as cancer, including metastasis, inflammatorydiseases, including arthritis, and diabetes. The anti-coagulant compoundis administered orally, parenterally e.g. by intravenous infusion (iv),intramuscular injection (im), subcutaneously (sc), or transdermally.

The specific dose of a compound administered according to this inventionto obtain therapeutic and/or prophylactic effects will, of course, bedetermined by the particular circumstances surrounding the case,including, for example, the compound administered, the rate ofadministration, the route of administration, and the condition beingtreated.

A typical daily dose for each of the above utilities is between about0.01 mg/kg and about 1000 mg/kg. The dose regimen may vary e.g. forprophylactic use a single daily dose may be administered or multipledoses such as 3 or 5 times daily may be appropriate. In critical caresituations a compound of the invention is administered by iv infusion ata rate between about 0.01 mg/kg/h and about 20 mg/kg/h and preferablybetween about 0.1 mg/kg/h and about 5 mg/kg/h.

The method of this invention also is practiced in conjunction with aclot lysing agent e.g. tissue plasminogen activator (t-PA), modifiedt-PA, streptokinase or urokinase. In cases when clot formation hasoccurred and an artery or vein is blocked, either partially or totally,a clot lysing agent is usually employed. A compound of the invention canbe administered prior to or along with the lysing agent or subsequent toits use, and preferably further is administered along with aspirin toprevent the reoccurrence of clot formation.

The method of this invention is also practiced in conjunction with aplatelet glycoprotein receptor (IIb/IIIa) antagonist, that inhibitsplatelet aggregation. A compound of the invention can be administeredprior to or along with the IIb/IIIa antagonist or subsequent to. its useto prevent the occurrence or reoccurrence of clot formation.

The method of this invention is also practiced in conjunction withaspirin. A compound of the invention can be administered prior to oralong with aspirin or subsequent to its use to prevent the occurrence orreoccurrence of clot formation. As stated above, preferably a compoundof the present invention is administered in conjunction with a clotlysing agent and aspirin.

This invention also provides pharmaceutical compositions for use in theabove described therapeutic method. Pharmaceutical compositions of theinvention comprise an effective antithrombotic amount of a compound offormula I in association with a pharmaceutically acceptable carrier,excipient or diluent. For oral administration the antithromboticcompound is formulated in gelatin capsules or tablets which may containexcipients such as binders, lubricants, disintegration agents and thelike. For parenteral administration the antithrombotic is formulated ina pharmaceutically acceptable diluent e.g. physiological saline (0.9percent), 5 percent dextrose, Ringer's solution and the like. Fortransdermal administration the antithrombotic is formulated in a patch.

The compound of the present invention can be formulated in unit dosageformulations comprising a dose between about 0.1 mg and about 1000 mg.Preferably the compound is in the form of a pharmaceutically acceptablesalt such as for example the sulfate salt, acetate salt or a phosphatesalt. An example of a unit dosage formulation comprises 5 mg of acompound of the present invention as a pharmaceutically acceptable saltin a 10 mL sterile glass ampoule. Another example of a unit dosageformulation comprises about 10 mg of a compound of the present inventionas a pharmaceutically acceptable salt in 20 mL of isotonic salinecontained in a sterile ampoule.

The antithrombotic compound can be administered by a variety of routesincluding oral, rectal, transdermal, subcutaneous, intravenous,intramuscular, and intranasal. The compound of the present inventionpreferably is formulated prior to administration. Another embodiment ofthe present invention is a pharmaceutical composition comprising aneffective amount of a compound of formula I or a pharmaceuticallyacceptable salt or solvate thereof in association with apharmaceutically acceptable carrier, diluent or excipient therefor.

The active ingredient in such formulations comprises from 0.1 percent to99.9 percent by weight of the formulation. By “pharmaceuticallyacceptable” it is meant the carrier, diluent or excipient must becompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

The present pharmaceutical compositions are prepared by known proceduresusing well known and readily available ingredients. The compositions ofthis invention may be formulated so as to provide quick, sustained, ordelayed release of the active ingredient after administration to thepatient by employing procedures well known in the art. In making thecompositions of the present invention, the active ingredient willusually be admixed with a carrier, or diluted by a carrier, or enclosedwithin a carrier which may be in the form of a capsule, sachet, paper orother container. When the carrier serves as a diluent, it may be asolid, semi-solid or liquid material which acts as a vehicle, excipientor medium for the active ingredient. Thus, the compositions can be inthe form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols, (as asolid or in a liquid medium), soft and hard gelatin capsules,suppositories, sterile injectable solutions, sterile packaged powders,and the like.

The following formulation examples are illustrative only and are notintended to limit the scope of the invention in any way. “Activeingredient,” of course, means a compound according to formula I or apharmaceutically acceptable salt or solvate thereof.

Formulation 1: Hard gelatin capsules are prepared using the followingingredients: Quantity (mg/capsule) Active ingredient 250 Starch, dried200 Magnesium stearate 10 Total 460 mg

Formulation 2: A tablet is prepared using the ingredients below:Quantity (mg/tablet) Active ingredient 250 Cellulose, microcrystalline400 Silicon dioxide, fumed 10 Stearic acid 5 Total 665 mg

The components are blended and compressed to form tablets each weighing665 mg.

Formulation 3: An aerosol solution is prepared containing the followingcomponents: Weight Active ingredient 0.25 Ethanol 25.75 Propellant 22(Chlorodifluoromethane) 70.00 Total 100.00

The active compound is mixed with ethanol and the mixture added to aportion of the propellant 22, cooled to 30° C. and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remainder of the propellant. The valveunits are then fitted to the container.

Formulation 4: Tablets, each containing 60 mg of active ingredient, aremade as follows: Active ingredient 60 mg Starch 45 mg Microcrystallinecellulose 35 mg Polyvinylpyrrolidone (as 10% solution in 4 mg water)Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5 mg Talc 1 mgTotal 150 mg

The active ingredient, starch and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The aqueous solution containingpolyvinylpyrrolidone is mixed with the resultant powder, and the mixturethen is passed through a No. 14 mesh U.S. sieve. The granules soproduced are dried at 50° C. and passed through a No. 18 mesh U.S.Sieve. The sodium carboxymethyl starch, magnesium stearate and talc,previously passed through a No. 60 mesh U.S. sieve, are then added tothe granules which, after mixing, are compressed on a tablet machine toyield tablets each weighing 150 mg.

Formulation 5: Capsules, each containing 80 mg of active ingredient, aremade as follows: Active ingredient 80 mg Starch 59 mg Microcrystallinecellulose 59 mg Magnesium stearate  2 mg Total 200 mg 

The active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 45 mesh U.S. sieve, and filled into hardgelatin capsules in 200 mg quantities.

Formulation 6: Suppositories, each containing 225 mg of activeingredient, are made as follows: Active ingredient   225 mg Saturatedfatty acid glycerides 2,000 mg Total 2,225 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

Formulation 7: Suspensions, each containing 50 mg of active ingredientper 5 mL dose, are made as follows: Active ingredient 50 mg Sodiumcarboxymethyl cellulose 50 mg Syrup 1.25 mL   Benzoic acid solution 0.10mL   Flavor q.v. Color q.v. Purified water to total  5 mL

The active ingredient is passed through a No. 45 mesh U.S. sieve andmixed with the sodium carboxymethyl cellulose and syrup to form a smoothpaste. The benzoic acid solution, flavor and color are diluted with aportion of the water and added, with stirring. Sufficient water is thenadded to produce the required volume.

Formulation 8: An intravenous formulation may be prepared as follows:Active ingredient 100 mg Isotonic saline 1,000 mL

The solution of the above ingredients generally is administeredintravenously to a subject at a rate of 1 mL per minute.

The ability of a compound of the present invention to be an effectiveand orally active antithrombotic agent is evaluated in one or more ofthe following assays.

The compound provided by the invention (formula I) selectively inhibitsthe action of thrombin and/or factor Xa in mammals. The inhibition isdemonstrated by in vitro inhibition of the amidase activity of theenzyme as measured in an assay in which the enzyme hydrolyzes achromogenic substrate. For example, thrombin hydrolyzesN-benzoyl-L-phenylalanyl-L-valyl-L-arginyl-p-nitroanilide,N-benzoyl-L-Phe-L-Val-L-Arg-p-nitroanilide.

The assay is carried out by mixing 50 μL buffer (0.03M Tris, 0.15M NaCl,pH 7.4) with 25 μL of human thrombin solution (purified human thrombin,Enzyme Research Laboratories, South Bend, Indiana, at 8 NIH units/mL)and 25 μL of test compound in a solvent (50% aqueous methanol (v:v)).Then 150 μL of an aqueous solution of the chromogenic substate (at 0.25mg/mL) are added and the rates of hydrolysis of the substrate aremeasured by monitoring the reactions at 405 nm for the release ofp-nitroaniline. Standard curves are constructed by plotting freethrombin concentration against hydrolysis rate. The hydrolysis ratesobserved with test compounds are then converted to “free thrombin”values in the respective assays by use of the standard curves. The boundthrombin (bound to test compound) is calculated by subtracting theamount of free thrombin observed in each assay from the known initialamount of thrombin used in the assay. The amount of free inhibitor ineach assay is calculated by subtracting the number of moles of boundthrombin from the number of moles of added inhibitor (test compound).

The Kass value is the hypothetical equilibrium constant for the reactionbetween thrombin and the test compound (I).${Thrombin} + {I\begin{matrix} \\\end{matrix}{Thrombin}\text{-}I}$$\quad {{Kass} = \frac{\left\lbrack {{Thrombin}\text{-}I} \right\rbrack}{\left\lbrack {({Thrombin}) \times (I)} \right\rbrack}}$

Kass is calculated for a range of concentrations of test compounds andthe mean value reported in units of liter per mole.

By substantially following the procedures described above for humanthrombin, and using other human blood coagulation system serineproteases and using fibrinolytic system serine proteases, with theappropriate chromogenic substrates, identified below, the selectivity ofthe compounds of the present invention with respect to the coagulationfactor serine proteases and to the fibronolytic serine proteases areevaluated as well as their substantial lack of interference with humanplasma clot fibrinolysis.

Human factors X, Xa, IXa, XIa, and XIIa are purchased from EnzymeResearch Laboratories, South Bend, Indiana; human urokinase from LeoPharmaceuticals, Denmark; and recombinant activated Protein C (aPC) isprepared at Eli Lilly and Co. substantially according to U.S. Pat. No.4,981,952. Chromogenic substrates:N-Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (for factor Xa);N-Cbz-D-Arg-Gly-Arg-p-nitroanilide (for factor IXa assay as the factorXa substrate); Pyroglutamyl-Pro-Arg-p-nitroanilide (for Factor XIa andfor aPC); H-D-Pro-Phe-Arg-p-nitroanilide (for factor XIIa); andPyroglutamyl-Gly-Arg-p-nitroanilide (for urokinase); are purchased fromKabi Vitrum, Stockholm, Sweden, or from Midwest Biotech, Fishers, Ind.Bovine trypsin is purchased from Worthington Biochemicals, Freehold,N.J., and human plasma kallikrein from Kabi Vitrum, Stockholm, Sweden.Chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide for plasmakallikrein is purchased from Kabi Vitrum, Stockholm, Sweden.N-Benzoyl-Phe-Val-Arg-p-nitroanilide, the substrate for human thrombinand for trypsin, is synthesized according to procedures described abovefor the compounds of the present invention, using known methods ofpeptide coupling from commercially available reactants, or purchasedfrom Midwest Biotech, Fishers, Ind.

Human plasmin is purchased from Boehringer Mannheim, Indianapolis, Ind.;nt-PA is purchased as single chain activity reference from AmericanDiagnostica, Greenwich, Connecticut; modified-t-PA6 (mt-PA6) is preparedat Eli Lilly and Company by procedure known in the art (See, Burck, etal., J. Biol. Chem., 265, 5120-5177 (1990). Plasmin chromogenicsubstrate H-D-Val-Leu-Lys-p-nitroanilide and tissue plasminogenactivator (t-PA) substrate H-D-Ile-Pro-Arg-p-nitroanilide are purchasedfrom Kabi Vitrum, Stockholm, Sweden.

In the chromogenic substrates described above the three-letter symbolsIle, Glu, Gly, Pro, Arg, Phe, Val, Leu and Lys are used to indicate thecorresponding amino acid group isoleucine, glutamic acid, glycine,proline, arginine, phenylalanine, valine, leucine and lysine,respectively.

The antithrombotic compound preferably should spare fibrinolysis inducedby urokinase, tissue plasminogen activator (t-PA) and steptokinase. Thiswould be important to the therapeutic use of such agents as an adjunctto streptokinase, t-PA or urokinase thrombolytic therapy and to the useof such agents as an endogenous fibrinolysis-sparing (with respect tot-PA and urokinase) antithrombotic agents. In addition to the lack ofinterference with the amidase activity of the fibrinolytic proteases,such fibrinolytic system sparing can be studied by the use of humanplasma clots and their lysis by the respective fibrinolytic plasminogenactivators.

Materials

Dog plasma is obtained from conscious mixed-breed hounds (either sexButler Farms, Clyde, N.Y., U.S.A.) by venipuncture into 3.8 percentcitrate. Fibrinogen is prepared from fresh dog plasma and humanfibrinogen is prepared from in-date ACD human blood at the fraction I-2according to previous procedures and specifications. Smith, Biochem. J.,185, 1-11 (1980); and Smith, et al., Biochemistry, 11, 2958-2967,(1972). Human fibrinogen (98 percent pure/plasmin free) is from AmericanDiagnostica, Greenwich, Connecticut. Radiolabeling of fibrinogen I-2preparations is performed as previously reported. Smith, et al.,Biochemistry, 11, 2958-2967, (1972). Urokinase is purchased form LeoPharmaceuticals, Denmark, as 2200 Ploug units/vial. Streptokinase ispurchased from Hoechst-Roussel Pharmaceuticals, Somerville, N.J.

Methods

Effects on Lysis of Human Plasma Clots by t-PA

Human plasma clots are formed in micro test tubes by adding 50 μLthrombin (73 NIH unit/mL) to 100 μL human plasma which contains 0.0229μCi 125-iodine labeled fibrinogen. Clot lysis is studied by overlayingthe clots with 50 μL of urokinase or streptokinase (50, 100, or 1000unit/mL) and incubating for 20 hours at room temperature. Afterincubation the tubes are centrifuged in a Beckman Microfuge. 25 μL 1 ofsupernate is added into 1.0 mL volume of 0.03 M tris/0.15 M NaCl bufferfor gamma counting. Counting controls 100 percent lysis are obtained byomitting thrombin (and substituting buffer). The antithrombotic compoundis evaluated for possible interference with fibrinolysis by includingthe compounds in the overlay solutions at 1, 5, and 10 μg/mLconcentrations. Rough approximations of IC₅₀ values are estimated bylinear extrapolations from data points to a value which would represent50 percent of lysis for that particular concentration of fibrinolyticagent.

ANTICOAGULANT ACTIVITY Materials

Dog plasma and rat plasma are obtained from conscious mixed-breed hounds(either sex, Butler Farms, Clyde, N.Y., U.S.A.) or from anesthetizedmale Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., Indianapolis,Ind., U.S.A.) by venipuncture into 3.8 percent citrate. Fibrinogen isprepared from in-date ACD human blood as the fraction I-2 according toprevious procedures and specifications. Smith, Biochem. J., 185, 1-11(1980); and Smith, et al., Biochemistry, 11, 2958-2967 (1972). Humanfibrinogen is also purchased as 98 percent pure/plasmin free fromAmerican Diagnostica, Greenwich, Conn. Coagulation reagents ACTIN,Thromboplastin, and Human plasma are from Baxter Healthcare Corp., DadeDivision, Miami, Fla. Bovine thrombin from Parke-Davis (Detroit, Mich.)is used for coagulation assays in plasma.

Methods

Anticoagulation Determinations

Coagulation assay procedures are as previously described. Smith, et al.,Thrombosis Research, 50, 163-174 (1988). A CoAScreener coagulationinstrument (American LABor, Inc.) is used for all coagulation assaymeasurements. The thrombin time (TT) is measured by adding 0.05 mLsaline and 0.05 mL thrombin (10 NIH units/mL) to 0.05 mL test plasma.The activated partial thromboplastin time (APTT) is measured byincubation of 0.05 mL test plasma with 0.05 mL Actin reagent for 120seconds followed by 0.05 mL CaCl₂ (0.02 M). The prothrombin time (PT) ismeasured by adding 0.05 mL saline and 0.05 mL Thromboplastin-C reagentto 0.05 mL test plasma. The compounds of formula I are added to human oranimal plasma over a wide range of concentrations to determineprolongation effects on the TT, APTT and PT assays. Linearextrapolations are performed to estimate the concentrations required todouble the clotting time for each assay.

Animals

Male Sprague Dawley rats (350-425 g, Harlan Sprague Dawley Inc.,Indianapolis, Ind.) are anesthetized with xylazine (20 mg/kg, s.c.) andketamine (120 mg/kg, s.c.) and maintained on a heated water blanket (37°C.). The jugular vein(s) is cannulated to allow for infusions.

Arterio-Venous Shunt Model

The left jugular vein and right carotid artery are cannulated with 20 cmlengths of polyethylene PE 60 tubing. A 6 cm center section of largertubing (PE 190) with a cotton thread (5 cm) in the lumen, is frictionfitted between the longer sections to complete the arterio-venous shuntcircuit. Blood is circulated through the shunt for 15 min before thethread is carefully removed and weighed. The weight of a wet thread issubtracted from the total weight of the thread and thrombus (see J. R.Smith, Br J Pharmacol, 77:29,1982).

FeCl₃ Model of Arterial Injury

The carotid arteries are isolated via a midline ventral cervicalincision. A thermocouple is placed under each artery and vesseltemperature is recorded continuously on a strip chart recorder. A cuffof tubing (0.058 ID×0.077 OD×4 mm, Baxter Med. Grade Silicone), cutlongitudinally, is placed around each carotid directly above thethermocouple. FeCl₃ hexahydrate is dissolved in water and theconcentration (20 percent) is expressed in terms of the actual weight ofFeCl₃ only. To injure the artery and induce thrombosis, 2.85 μL ispipetted into the cuff to bathe the artery above the thermocouple probe.Arterial occlusion is indicated by a rapid drop in temperature. The timeto occlusion is reported in minutes and represents the elapsed timebetween application of FeCl₃ and the rapid drop in vessel temperature(see K. D. Kurz, Thromb. Res., 60:269,1990).

Arterio-Venous (AV) Shunt Model in the Rabbit

Efficacy and potency of a compound are studied in a model of thrombosisin the anesthetized rabbit in which blood is shunted from the carotidartery through tubing to the left jugular vein. The shunt houses athread upon which thrombotic material is deposited and is quantified inthe presence and absence of a test compound. Specifically, the leftjugular vein and right carotid artery are cannulated (PE 200 tubing, 22cm). The tip of the arterial tubing is advanced approximately 1 cm andthe tip of the venous tubing is advanced approximately 1 cm. Thearterial and venous shunt segments are friction fitted into a centersection of larger tubing (PE 260, 6 cm) with 4 strands of cotton thread(5.5 cm each), knotted together with a single knot at the end, in thelumen. Blood is circulated through the shunt for 15 min. The arterialand venous segments are clamped, the center section is detached, heldvertically and the thread is carefully removed and weighed. The weightof a wet thread (38 mg, average of 10-4-stranded identical lengths) issubtracted from the total weight of the thread and thrombus. A drug isinfused through a second catheter in the right jugular vein (2 cm insidevessel) starting 15 min before and throughout the 15 min period of bloodcirculation through the shunt. Another catheter, for sampling blood alsois implanted in the right jugular and the tip is advanced 15 cm toensure it is distal to the heart such that the sample is not enrichedwith blood flowing past the tip of the drug infusion catheter.

Spontaneous Thrombolysis Model

In vitro data suggests that peptide thrombin or factor Xa inhibitors athigher concentration may inhibit other serine proteases, such as plasminand tissue plasminogen activator. To assess if a compound inhibitsfibrinolysis in vivo, the rate of spontaneous thrombolysis is determinedby implanting a labeled whole blood clot into the pulmonary circulation.Rat blood (1 mL) is mixed rapidly with bovine thrombin (4 IU, ParkeDavis) and ¹²⁵I human Fibrogen (5 μCi, ICN), immediately drawn intosilastic tubing and incubated at 37° C. for 1 hour. The aged thrombus isexpelled from the tubing, cut into 1 cm segments, washed 3× in normalsaline and each segment is counted in a gamma counter. A segment withknown counts is aspirated into a catheter that is subsequently implantedinto the jugular vein. The catheter tip is advanced to the vicinity ofthe right atrium and the clot is expelled to float into the pulmonarycirculation. One hour after implant, the heart and lungs are harvestedand counted separately. Thrombolysis is expressed as a percentage where:${\% \quad {Thrombolysis}} = {\frac{\left( {{{injected}\quad {cpm}} - {{lung}\quad {cpm}}} \right)}{{injected}\quad {cpm}} \times 100}$

The fibrinolytic dissolution of the implanted clot occurstime-dependently (see J. P. Clozel, Cardiovas. Pharmacol., 12:520,1988).

Coagulation Parameters

Plasma thrombin time (TT) and activated partial thromboplastin time(APTT) are measured with a fibrometer. Blood is sampled from a jugularcatheter and collected in syringe containing sodium citrate (3.8percent, 1 part to 9 parts blood). To measure TT, rat plasma is mixedwith saline and human thrombin at 37° C. For.APTT, plasma (0.1 mL) andAPTT solution (0.1 mL, Organon Teknika) are incubated for 5 minutes (37°C.) and CaCl₂ (0.1 mL, 0.025 M) is added to start coagulation. Assaysare done in duplicate and averaged.

Index of Bioavailability

A measure of bioactivity, plasma thrombin time (TT), serves as asubstitute for the assay of parent compound on the assumption thatincrements in TT resulted from thrombin inhibition by parent only. Thetime course of the effect of the thrombin inhibitor upon TT isdetermined after i.v bolus administration to anesthetized rats and afteroral treatment of fasted conscious rats. Due to limitations of bloodvolume and the number of points required to determine the time coursefrom time of treatment to the time when the response returns topretreatment values, two populations of rats are used. Each samplepopulation represents alternating sequential time points. The average TTover the time course is used to calculate area under the curve (AUC).The index of bioavailability is calculated by the formula shown belowand is expressed as percent relative activity.

The area under the curve (AUC) of the plasma TT time course isdetermined and adjusted for the dose. This index of bioavailability istermed “% Relative Activity” and is calculated as${\% \quad {Relative}\quad {Activity}} = {\frac{{AUC}\quad {po}}{{AUC}\quad {iv}} \times \frac{{Dose}\quad {iv}}{{Dose}\quad {po}} \times 100}$

Compounds

Compound solutions are prepared fresh daily in normal saline and areinjected as a bolus or are infused starting 15 minutes before andcontinuing throughout the experimental perturbation which is 15 minutesin the arteriovenous shunt model and 60 minutes in the FeCl₃ model ofarterial injury and in the spontaneous thrombolysis model. Bolusinjection volume is 1 mL/kg for i.v., and 5 mL/kg for p.o. and infusionvolume is 3 mL/hr.

Statistics

Results are expressed as means+/−SEM. One-way analysis of variance isused to detect statistically significant differences and then Dunnett'stest is applied to determine which means are different. Significancelevel for rejection of the null hypothesis of equal means is P<0.05.

Animals

Male dogs (Beagles; 18 months—2 years; 12-13 kg, Marshall Farms, NorthRose, N.Y. 14516) are fasted overnight and fed Purina certifiedPrescription Diet (Purina Mills, St. Louis, Mo.) 240 minutes afterdosing. Water is available ad libitum. The room temperature ismaintained between 66-74° F.; 45-50 percent relative humidity; andlighted from 0600-1800 hours.

Pharmacokinetic Model

Test compound is formulated immediately prior to dosing by dissolving insterile 0.9 percent saline to a 5 mg/mL preparation. Dogs are given asingle 2 mg/kg dose of test compound by oral gavage. Blood samples (4.5mL) are taken from the cephalic vein at 0.25, 0.5, 0.75, 1, 2, 3, 4 and6 hours after dosing. Samples are collected in citrated Vacutainer tubesand kept on ice prior to reduction to plasma by centrifugation. Plasmasamples are analyzed by HPLC MS. Plasma concentration of test compoundis recorded and used to calculate the pharmacokinetic parameters:elimination rate constant, Ke; total clearance, Clt; volume ofdistribution, V_(D); time of maximum plasma test compound concentration,Tmax; maximum concentration of test compound of Tmax, Cmax; plasmahalf-life, t0.5; and area under the curve, A.U.C.; fraction of testcompound absorbed, F.

Canine Model of Coronary Artery Thrombosis

Surgical preparation and instrumentation of the dogs are as described inJackson, et al., Circulation, 82, 930-940 (1990). Mixed-breed hounds(aged 6-7 months, either sex, Butler Farms, Clyde, N.Y., U.S.A.) areanesthetized with sodium pentobarbital (30 mg/kg intravenously, i.v.),intubated, and ventilated with room air. Tidal volume and respiratoryrates are adjusted to maintain blood PO₂, PCO₂, and pH within normallimits. Subdermal needle electrodes are inserted for the recording of alead II ECG.

The left jugular vein and common carotid artery are isolated through aleft mediolateral neck incision. Arterial blood pressure (ABP) ismeasured continuously with a precalibrated Millar transducer (model(MPC-500, Millar Instruments, Houston, Tex., U.S.A.) inserted into thecarotid artery. The jugular vein is cannulated for blood sampling duringthe experiment. In addition, the femoral veins of both hindlegs arecannulated for administration of test compound.

A left thoracotomy is performed at the fifth intercostal space, and theheart is suspended in a pericardial cradle. A 1- to 2-cm segment of theleft circumflex coronary artery (LCX) is isolated proximal to the firstmajor diagonal ventricular branch. A 26-gauge needle-tipped wire anodalelectrode (Teflon-coated, 30-gauge silverplated copper wire) 3-4 mm longis inserted into the LCX and placed in contact with the intimal surfaceof the artery (confirmed at the end of the experiment). The stimulatingcircuit is completed by placing the cathode in a subcutaneous (s.c.)site. An adjustable plastic occluder is placed around the LCX, over theregion of the electrode. A precalibrated electromagnetic flow probe(Carolina Medical Electronics, King, N.C., U.S.A.) is placed around theLCX proximal to the anode for measurement of coronary blood flow (CBF).The occluder is adjusted to produce a 40-50 percent inhibition of thehyperemic blood flow response observed after 10-s mechanical occlusionof the LCX. All hemodynamic and ECG measurements are recorded andanalyzed with a data acquisition system (model M3000, ModularInstruments, Malvern, Pa. U.S.A.).

Thrombus Formation and Compound Administration Regimens

Electrolytic injury of the intima of the LCX is produced by applying100-μA direct current (DC) to the anode. The current is maintained for60 min and then discontinued whether the vessel has occluded or not.Thrombus formation proceeds spontaneously until the LCX is totallyoccluded (determined as zero CBF and an increase in the S-T segment).Compound administration is started after the occluding thrombus isallowed to age for 1 hour. A 2-hour infusion of the compounds of thepresent invention at doses of 0.25, 0.5 and 1 mg/kg/hour is begunsimultaneously with an infusion of thrombolytic agent (e.g. tissueplasminogen activator, streptokinase, APSAC). Reperfusion is followedfor 3 hour after administration of test compound. Reocclusion ofcoronary arteries after successful thrombolysis is defined as zero CBFwhich persisted for at least 30 minutes.

Hematology and Template Bleeding Time Determinations

Whole blood cell counts, hemoglobin, and hematocrit values aredetermined on a 40-μL sample of citrated (3.8 percent) blood (1 partcitrate:9 parts blood) with a hematology analyzer (Cell-Dyn 900,Sequoia-Turner. Mount View, Calif., U.S.A.). Gingival template bleedingtimes are determined with a Simplate II bleeding time device (OrganonTeknika Durham, N.C., U.S.A.). The device is used to make 2 horizontalincisions in the gingiva of either the upper or lower left jaw of thedog. Each incision is 3 mm wide×2 mm deep. The incisions are made, and astopwatch is used to determine how long bleeding occurs. A cotton swabis used to soak up the blood as it oozes from the incision. Templatebleeding time is the time from incision to stoppage of bleeding.Bleeding times are taken just before administration of test compound (0min), 60 min into infusion, at conclusion of administration of the testcompound (120 min), and at the end of the experiment.

All data are analyzed by one-way analysis of variance (ANOVA) followedby Student-Neuman-Kuels post hoc t test to determine the level ofsignificance. Repeated-measures ANOVA are used to determine significantdifferences between time points during the experiments. Values aredetermined to be statistically different at least at the level ofp<0.05. All values are mean±SEM. All studies are conducted in accordancewith the guiding principles of the American Physiological Society.Further details regarding the procedures are described in Jackson, etal., J. Cardiovasc. Pharmacol., 21, 587-599 (1993).

The compound of formula Ia has particularly surprising properties. Thus,as DL-Arg-Ia (an approximately equal mixture of epimers at the Argcenter, see Example 1, below), the compound demonstrated similar highpotency as an inhibitor of thrombin (Kass=352 million, average of 3assays) and of factor Xa (Kass=301 million, average of 3 assays).Moreover, highly potent antithrombotic efficacy was shown in the rabbitAV shunt model where an ED50% (infusion dose to reduce thrombus weightby 50%) value of 0.008 mg/kg/h i.v. was observed. At this dose, therewas no observed change in the thrombin time ratio.

The following Examples are provided to further describe the inventionand are not to be construed as limitations thereof. The abbreviationsused in the examples have the following meanings.

Amino acids: Azt=azetidine-2-carboxylic acid,

Pro=proline

Anal.=elemental analysis

Boc=t-butyloxycarbonyl

Bn=benzyl

t-Bu=t-butyl

n-BuLi=butyllithium

Cbz=benzyloxycarbonyl

DCC=dicyclohexylcarbodiimide

DMF=dimethylformamide

DMSO=dimethylsulfoxide

Et=ethyl

EtOAc=ethyl acetate

Et₂O=diethyl ether

EtOH=ethanol

FAB-MS=fast atom bombardment mass spectrum

FD-MS=field desorption mass spectrum

HPLC=High Performance Liquid Chromatography

HRMS=high resolution mass spectrum

HOBT=1-hydroxybenzotriazole hydrate

i-PrOH=isopropanol

IR=Infrared Spectrum

Me=methyl

MeOH=methanol

NMR=Nuclear Magnetic Resonance

RPHPLC=Reversed Phase High Performance Liquid Chromatography

SiO₂=silica gel

TEA=triethylamine

TFA=trifluoroacetic acid

THF=tetrahydrofuran

TLC=thin layer chromatography

Ts=tosyl (p-toluenesulfonyl)

Unless otherwise stated, pH adjustments and work up are with aqueousacid or base solutions.

The R_(f) values in the examples are determined by silica gel thin layerchromatography (Kieselgel 60 F-254) in the following systems:

(A) Chloroform-Methanol-Acetic Acid 135:15:1

(B) Ethyl Acetate-Acetic Acid-Absolute Alcohol 90:10:10

(C) Ethyl Acetate-Hexanes 30:70

EXAMPLE 1 Preparation of(1R,4aR,8aR)-Perhydroisoquinolin-1-yl-carbonyl-N-[(1S)-4-[(aminoiminomethyl)amino]-1-(benzo-thiazol-2-ylcarbonyl)butyl]-L-prolinamide.1.5 H₂SO₄

A. N-Methoxycarbonylphenethylamine

To a stirred solution of phenethylamine (75.2 mL, 0.6 mol) andtriethylamine (83 mL, 0.6 mol) in THF (500 mL) was added slowly methylchloroformate (46.2 mL, 0.6 mol) dissolved in THF (50 mL). After thereaction was stirred for an additional 1 h at room temperature, diethylether (2 L) and 1 N HC1 (800 mL) was added. The organic layer was washedwith water, dried (MgSO₄), and concentrated in vacuo to give a clear oilof pure title compound (102 g, 95%).

B. 2-Methoxycarbonyl-DL-1,2,3,4-tetrahydoisoquinoline-1-carboxylic acid

To a solution of N-methoxycarbonyl phenethylamine (102 g, 0.57 mol) intrifluoroacetic acid (300 mL) was added glyoxylic acid (63 g, 0.68 mol),and the mixture was heated to reflux temperature. After 4 h at refluxthe reaction was cooled to room temperature, solvent removed in vacuo,and diethyl ether (800 mL)/water (100 mL) was added to the residue. Thereaction mixture pH was raised to 12 with 5 N NaOH and the aqueous layerseparated. To the aqueous layer was added diethyl ether (500 mL), andthe solution was acidified to pH 2.5 with 5 N HC1. The organic layer wasseparated, dried (MgSO₄), filtered, and the filtrate was concentrated invacuo to afford an oil of pure title compound (107 g, 80%); FAB-MS 236(MH⁺).

C. 2-Methoxycarbonyl-DL-1,2,3,4-tetrahydoisoquinoline-1-carboxylic acidt-butyl ester

To a stirred, cooled (0° C.), solution of2-methoxy-carbonyl-DL-1,2,3,4-tetrahydoisoquinoline-1-carboxylic acid(105 g, 0.45 mol) in CH₂Cl₂ (200 mL) was added t-butanol (52 mL, 0.54mol) and DCC (92 g, 0.45 mol). After 2 h at 0° C. and 24 h at roomtemperature the solvent was removed in vacuo, and ethyl acetate (800mL)/1 N NaHCO₃ (300 mL) was added to the residue. The organic layer wasseparated, washed with water, 1.5 N citric acid, and water. The organiclayer was dried (MgSO₄), filtered, and the filtrate was concentrated invacuo to afford an oil of pure title compound (106 g, 81%); FAB-MS 292(MH⁺); TLC R_(f) (A) 0.61; elemental analysis (calcd) C₁₆H₂₁NO₄: C,65.96; H, 7.27; N, 4.81; Found: C, 66.24, H, 7.28, N, 4.73.

D. 2-methoxycarbonyl-(1RS,4aSR,8aSR)-perhydroisoquinoline-1-carboxylicacid t-butyl ester

A solution of2-methoxycarbonyl-DL-1,2,3,4-tetrahydro-isoquinoline-1-carboxylic acidt-butyl ester (105 g, 0.36 mol) in t-butanol (800 mL) was reduced over5% Rh/Al₂O₃ (52.5 g) at 55 bar (800 psi) of hydrogen in a high pressureapparatus at 50° C. for 24 hours. The reaction mixture was filteredthrough a pad of diatomaceous earth, and the filtrate was concentratedin vacuo. The resulting oil was dried to give pure title compound (96.5g, 90%); FD-MS 298 (MH⁺); TLC R_(f) (C) 0.63.

E. 2-Methoxycarbonyl-(1RS,4aRS,8aRS)-perhydroisoquinoline-1-carboxylicacid ethyl ester

To a solution of2-methoxycarbonyl-(1RS,4aSR,8aSR)-perhydroisoquinoline-1-carboxylic acidt-butyl ester (81.2 g, 273 mmol) in EtOH (500 mL) was added sodiumethoxide (21% in ethanol) (88.4 mL, 273 mmol) and the reaction mixturewas refluxed (24 h). The organic solvent was evaporated in vacuo, ethylacetate (400 mL) and water (100 mL) was added to the residue. Theorganic layer was separated, washed twice with water, dried (MgSO₄),filtered, and the filtrate was concentrated in vacuo to afford an oil ofpure title compound (70 g,.95%); FAB-MS 270 (MH⁺); TLC R_(f) (A) 0.61.

F. 2-Methoxycarbonyl-(1RS,4aRS,8aRS)-perhydroisoquinoline-1-carboxylicacid

To a solution of the product of step E (70 g, 260 mmol) in THF (250 mL)was added 2 N NaOH (156 mL, 312 mmol) and the reaction mixture stirredat room temperature (30 h). The organic solvent was evaporated in vacuo,diethyl ether (400 mL) and water (100 mL) was added to the residue. Theaqueous layer separated and ethyl acetate (400 mL) was added. The pH ofthe solution was adjusted to 2.0 with 5 N HCl. The organic layer wasdried (MgSO₄), filtered, and the filtrate was concentrated in vacuo togive a clear oil. The oil was crystallized from hexane (200 mL) toafford pure title compound (46.4 g,.74%); FAB-MS 242 (MH⁺); TLC R_(f)(A) 0.36; elemental analysis (calcd) C₁₂H₁₉NO₄: C, 59.74; H, 7.94; N,5.81; Found: C, 59.95, H, 7.88, N, 5.54. NMR assignments were made byhomonuclear decoupling, COSY, HMQC, and DEPT experiments.

G. 2-Cbz-(1RS,4aRS,8aRS)-perhydroisoquinoline-1-carboxylic acid

To a stirred solution of the product of step F (46 g, 191 mmol), at roomtemperature, in anhydrous CH₃CN (200 mL) under an inert atmosphere wasadded a solution of iodotrimethylsilane (62.4 mL, 440 mmol) in CH₃CN (60mL). The reaction mixture was stirred at 55° C. for 30 min and cooled toroom temperature. The reaction was quenched with water (100 mL) followedby sodium metabisulfite (1 g). The pH of the reaction was raised to 10.0with 5 N NaOH, and benzyl chloroformate (27.3 mL, 191 mmol) was addeddropwise while the pH maintained at 10 with 2 N NaOH. After the reactionwas stirred for an additional 30 min at room temperature, the organicsolvent was evaporated in vacuo, and diethyl ether (200 mL) was added.The reaction was allowed to stand at room temperature (2 h) and ethylacetate (200 mL) was added. The aqueous solution was acidified to pH 2.5with 5 N HCl; the organic layer was separated, dried (MgSO₄), filtered,and the filtrate was concentrated in vacuo to give pure title compoundas an oil (39.5 g, 65%); FAB-MS 318 (MH⁺); elemental analysis (calcd)C₁₈H₂₃NO₄: C, 68.12; H, 7.30; N, 4.41; Found: C, 66.37, H, 7.52, N,4.37.

H. 2-Cbz-(1RS,4aRS,8aRS)-perhydroisoquinoline-1-carbonyl-Pro-O-t-Bu

To a stirred, cooled (0° C.) solution of the product of step G (39 g,123 mmol) in DMF (200 mL) was added proline t-butyl ester (21.1 g, 123mmol), 1-hydroxybenzotriazole (16.6 g, 123 mmol), and DCC (25.3 g, 123mmol). The reaction mixture was stirred for 2 h at 0° C. and 24 h atroom temperature. The reaction precipitate was filtered and the filtrateconcentrated in vacuo to an oil. The oil was dissolved in EtOAc (200 mL)and water (100 mL). The organic layer was washed sequentially with 1 NNaHCO₃, water, 1.5 N citric acid, and water. The organic layer was dried(MgSO₄), filtered, and the filtrate evaporated to an amorphous solid ofthe title compound as a mixture of diastereomers (52.7 g, 91%) FAB-MS471 (MH⁺).

I. 2-Cbz-(4aR,8aR)-perhydroisoquinoline-1(R)-carbonyl-Pro-OH

To a stirred solution of the product of step H (52.4 g, 111 mmol) inCH₂Cl₂ (20 mL) was added trifluoroacetic acid (70 mL) and anisole (5mL). The reaction mixture was stirred at room temperature for 1 h andconcentrated in vacuo without heating. The residue was diluted withdiethyl ether (400 mL), water (100 mL), and the pH of the solution wasadjusted to 10.0 with 5 N NaOH. The aqueous layer separated and ethylacetate (300 mL) was added. The pH of the solution was adjusted to 2.5with 5 N HCl; the organic layer was separated, dried (MgSO₄), filtered,and the filtrate was concentrated in vacuo to give a clear oil. The oilwas dissolved in diethyl ether (500 mL) and(L)-(−)-alpha-methylbenzylamine was added to the solution. The solutionwas allowed to stand at room temperature (24 h). The resulting solid wasfiltered, washed with diethyl ether and dried. The solid was suspendedin ethyl acetate, washed with 1.5 N citric acid, and water. The organiclayer was dried (MgSO₄), filtered, and the filtrate evaporated to givethe title compound as an oil (20.2 g, 44%). FAB-MS 415 (MH⁺); [a]D=3.2°(C=0.5, MeOH); elemental analysis (calcd) C₂₃H₃₀N₂O₅: C, 66.65; H, 7.30;N, 6.76. Found: C, 66.38, H, 7.36, N, 6.63.

J. Boc-Arg(Tosyl)-N(OMe)Me

To a stirred, cooled (0° C.) solution of Boc-L-Arg(Tosyl)-OH (34.2 g,79.8 mmol) in DMF (150 mL) was added N,O-dimethylhydroxylaminehydrochloride (15.6 g, 159.6 mmol) followed by diisopropylethylamine(27.8 mL, 159.6 mmol), HOBT (10.8 g, 79.8 mmol), and DCC (16.5 g, 79.8mmol). The reaction mixture was stirred for h at 0° C. and warmed toroom temperature and stirred 18 h. The reaction mixture was cooled (0°C.), the precipitate filtered, and the mother liquor concentrated todryness in vacuo. The resulting oil was dissolved in EtOAc and waswashed sequentially with 1 N NaHCO₃, water, 1.5 N citric acid, andwater. The organic solution was dried (MgSO₄), and evaporated to drynessin vacuo. The resulting oil was dissolved in EtOAc; and, after standingat 4° C. (4 h), the precipitate was filtered, washed with EtOAc anddried to give pure title compound (21.2 g, 57%). FAB-MS 472 (MH⁺); Anal.Calcd. for C₂₀H₃₃N₅O₆S: C, 50.94; H, 7.05; N, 14.85. Found: C, 52.29; H,7.32; N, 14.98.

K. 2-[Boc-Arg(Tosyl)]benzothiazole

To a stirred, cooled (−78° C.), solution of anhydrous THF (150 mL) wasadded 1.6 M n-butyllithium in hexane (150 mL, 240 mmol) under an inertatmosphere. To the reaction mixture was slowly added a solution ofbenzothiazole (32.4 g, 240 mmol) in THF (150 mL). After addition, asolution of the amide of step J (22.6 g, 48 mmol) in DMSO (27 mL) wasadded slowly to the reaction mixture. The reaction mixture was stirredat −78° C. for 3 h and for 40 min with an ice bath. The reaction mixturewas diluted with 1.5 N citric acid (300 mL) slowly. The resultingmixture was concentrated in vacuo to approximately 400 mL before EtOAc(200 mL) and water (100 mL) were added. The organic layer was washed3×with water, dried (MgSO₄), and evaporated in vacuo to give an oil. Thecrude oil was purified by chromatography on silica gel using a stepgradient elution (CHCl₃100 to EtOAc 100) to yield pure ketone (17.4 g,66%). FAB-MS 546 (MH⁺); [α]D=25.8° (C, 0.5 CHCl₃); elemental analysis(calcd) C₂₅H₃₁N₅O₅S: C, 55.03; H, 5.73; N, 12.83. Found: C, 54.73, H,5.72, N, 12.69.

L. 2-[Boc-Arg(Tosyl)(OH)]benzothiazole

To a stirred, cooled (0° C.) solution of the ketone of Part K (2.7 g,4.95 mmol) in EtOH (60 mL) was added sodium borohydride (187 mg, 4.95mmol). The reaction mixture was stirred at 0° C. for 1 h and slowlywarmed to room temperature overnight. The mixture's pH was adjusted to2.0 with 2 N HCl and then raised to pH 7.0 with 1 N NaOH. The resultingmixture was diluted with EtOAc (200 mL) and water (100 mL). The organiclayer was dried (MgSO₄), and evaporated to give the crude alcohol (2.7g, 99%). FAB-MS 548 (MH⁺); TLC R_(f) (A) 0.21.

M.Benzyloxycarbonyl-(1R,4aR,8aR)-perhydroisoquinolin-1-ylcarbonyl-N-[(1S)-4[(N-tosylaminoiminomethyl)amino]-1-[(benzothiazol-2-yl)(hydroxy)methyl]butyl]-L-prolinamide

To a flask containing the alcohol of step L (2.6 g, 4.75 mmol) was addedanisole (5 mL), followed by trifluoroacetic acid (70 mL). The reactionmixture was stirred at 0° C. for 20 min and concentrated in vacuowithout heating. The reaction mixture was diluted with diethyl ether(400 mL), and the solid was filtered and dried to give 2.3 grams ofcrude 2-[H-Arg(Tosyl)(OH)]benzothiazole.

In flask 1 the product of step I (1.97 g, 4.75 mmole) was dissolved inCH₂Cl₂ (50 mL), cooled to −15° C., and N-methylmorpholine (0.52 mL, 4.75mmole) was added, followed by isobutyl chloroformate (0.62 mL, 4.75mmole). The reaction mixture was stirred at −15° C. for 2 min.

In flask 2 the above crude 2-[H-Arg(Tosyl)(OH)]-benzothiazole (2.3 g,4.74 mmole) was dissolved in CH₂Cl₂ (20 mL), cooled to 0° C., anddiisopropylethylamine (2.5 mL, 14.3 mmole) was added to the solution.The reaction mixture was stirred at 0° C. for 2 min.

The contents of flask 2 was added to flask 1, and the reaction mixturewas stirred for 2 h (−15° C.) followed by 24 h at room temperature. Tothe reaction mixture was added 1 N NaHCO₃ (1 mL) and the reactionsolvent was removed in vacuo to afford an oil. The residue was dissolvedin EtOAc (200 mL) and washed sequentially with 1.5 N citric acid, water,1 N NaHCO₃ (100 mL), and water. The organic solution was dried (MgSO₄),and concentrated to dryness in vacuo to give the title alcohol.(9.0 g,39%) as a crude solid. FAB-MS 844 (MH⁺); TLC R_(f) (A) 0.34.

N.Benzyloxycarbonyl-(1R,4aR,8aR)-perhydro-isoquinolin-1-ylcarbonyl-N-[(1S)-4-[(aminoiminomethyl)-amino]-1-[(benzothiazol-2-ylcarbonyl)butyl]-L-prolinamide

A 500 mL three necked flask, equipped with a magnetic stirring bar and athermometer, and under nitrogen, was charged with a solution of oxalylchloride (0.74 mL, 8.4 mmol) in 10 mL of anhydrous CH₂Cl₂ and placed ina dry ice-acetone bath (−55° C. internal temperature). A solution ofDMSO (1.2 mL, 17 mmol) in 200 mL of CH₂Cl₂ was added at a rate to keepthe internal temperature at −55° C. Stirring was continued for 5 minutesand a solution of the alcohol of step M (1.4 g, 1.66 mmol) in 10 mLCH₂Cl₂ was added in one portion. The mixture was allowed to warm to −10°C. and stirred for 40 minutes. The solution was cooled (−55° C.),triethylamine (3.9 mL, 28 mmol) was added slowly, and, after addition,the cooling bath removed. When the temperature reached −20° C. a 1.5 Ncitric acid solution was added to the reaction. The organic layer wasseparated, washed once with water, dried (MgSO₄), and concentrated todryness in vacuo to give the title protected ketone (1.4 g, 100%). FABMS m/z 842 (MH⁺); elemental analysis (calcd) C₄₃H₅₁N₇O₇S₂: C, 61.34; H,6.10; N, 11.64. Found: C, 61.66, H, 6.58, N, 10.52.

O.(1R,4aR,8aR)-Perhydroisoquinolin-1-ylcarbonyl-N-[(1S)-4-[(aminoiminomethyl)amino]-1-(benzothiazol-2-ylcarbonyl)-butyl]-L-prolinamide.1.5 H₂SO₄

The reaction flask of a HF-Reaction apparatus was charged with theprotected ketone of step N (1.35 g, 1.60 mmol) and applied to HFdeprotection of all protecting groups. After treating the peptide with10 mL HF containing 1.0 mL of anisole and 1.0 mL dimethylsulfide for 1 hat 0° C., the HF was evaporated. The residue was treated with diethylether, and the precipitate was filtered and dried to a white solid. Thesolid (0.9 g) was dissolved in 0.01% H₂SO₄ and applied to a 5×25 cm ofcolumn Vydac C₁₈ resin. A gradient of increasing concentrations of CH₃CN(2% to 35%) was used to elute the peptide from the column. Fractionswere collected and pooled on the basis of analytical RP-HPLC profile.The combined fractions were adjusted to pH 4.2 using AG1-X8 resin(Bio-Rad analytical anion exchange resin 50-100 mesh) in hydroxide form.The solution was filtered, and the filtrate was lyophilized to affordpure title compound (0.403 g, 36%). FAB-MS 554 (MH⁺); elemental analysis(calcd) C₂₈H₃₉N₇O₃S. 1.5H₂SO₄: C, 47.99; H, 6.04; N, 13.99. Found: C,48.02, H, 6.02, N, 13.33.

Examination of the above compound by RPHPLC (C₁₈ resin, gradientelution, 5-45% acetonitrile containing 0.1% TFA —water containing 0.1%TFA) revealed the product to be a mixture of about 95:5 S:R-isomers atthe Arg center. This compound may be denoted as L-Arg-Ia forconvenience.

(1R,4aR,8aR)-Perhydroisoquinolin-1-ylcarbonyl-N-[(1RS)-4-[(aminoiminomethyl)amino]-1-(benzothiazol-2-ylcarbonyl)-butyl]-L-prolinamide.1.5 H₂SO₄

In a different preparation of this compound, in which a larger excess ofthe 2-benzothiazolyllithium reagent was used in the step correspondingto step K, above, a final product was obtained with an S:R-isomer ratioof about 1:1 as measured by RPHPLC and ¹³C NMR. This compound may bedenoted as DL-Arg-Ia for convenience.

EXAMPLE 2 Preparation of(1R,4aS,8aS)-Perhydroisoquinolin-1-yl-carbonyl-N-[(1S)-4-[(aminoiminomethyl)amino]-1-(benzo-thiazol-2-ylcarbonyl)butyl]-L-prolinamide.H₂SO₄

(I.e., insert the missing nitrogen atom in the perhydroisoquinolinering.)

Using the isomeric2-Cbz-(4aS,8aS)-perhydro-isoquinoline-1(R)-carbonyl-Pro-OH andprocedures similar to those described above, the title compound wasobtained. FAB-MS 554 (MH⁺); elemental analysis (calcd)C₂₈H₃₉N₇O₃S.H₂SO₄.H₂O: C, 50.21; H, 6.47; N, 14.64. Found: C, 50.12, H,6.06, N, 14.42; [a]D=−69.60° (C, 0.5 MeOH).

What is claimed is:
 1. A compound of formula I Y—CO—X—CO—Arg—R  Iwherein Arg is L-arginyl; R is 2-benzothiazolyl; X—CO— is L-prolyl or(S)-azetidin-2-carbonyl; and Y—CO— is a group of formula IIa, IIb, IIcor IId;

or a pharmaceutically acceptable salt thereof.
 2. The compound (or saltthereof) of claim 1 in which X—CO— is L-prolyl (Pro).
 3. The compound(or salt thereof) of claim 2 in which X—CO— is Pro and Y—CO— is a groupof formula IIa or IIb.
 4. The compound (or salt thereof) of claim 3 inwhich X—CO— is Pro and Y—CO— is a group of formula IIa.
 5. The compound(or salt thereof) of claim 3 in which X—CO— is Pro and Y—CO— is a groupof formula IIb.
 6. The compound (or salt thereof) of any one of claims1-5 wherein the chiral center indicated by the asterisk in the group Yis of (R)-stereochemistry corresponding to that of a D-amino acid. 7.The compound as claimed in claim 1 which is(1R,4aR,8aR)-perhydroisoquinolin-1-ylcarbonyl-N-[(1S)-4-[(aminoiminomethyl)amino]-1-(benzothiazol-2-ylcarbonyl)-butyl]-L-prolinamide,or a pharmaceutically acceptable salt thereof.
 8. The compound asclaimed in claim 1 which is(1R,4aR,8aR)-perhydroisoquinolin-1-ylcarbonyl-N-[(1RS)-4-[(aminoiminomethyl)amino]-1-(benzothiazol-2-ylcarbonyl)-butyl]-L-prolinamide,or a pharmaceutically acceptable salt thereof.
 9. The salt as claimed inclaim 7 or 8 which is the acid addition salt with sulfuric acid.
 10. Apharmaceutical composition comprising a compound of formula I as claimedin claim 1, or a pharmaceutically acceptable salt thereof, inassociation with a pharmaceutically acceptable carrier, diluent orexcipient.
 11. A process for preparing a compound of formula I (or apharmaceutically acceptable salt thereof) as provided in claim 1 whichis oxidation of the alcohol of a corresponding compound of formula IIIY—CO—X—CO—Arg(OH)—R  III in which Arg(OH) indicates that the carbonylportion of the L-arginyl group is replaced by a hydroxymethylene group;whereafter, when a functional group is protected using a protectinggroup, removing the protecting group; and whereafter, when apharmaceutically acceptable salt of a compound of formula I is required,it is obtained by reacting the basic form of the compound of formula Iwith an acid affording a physiologically acceptable counterion or by anyother conventional procedure; and wherein Arg, R, X and Y are defined asin claim
 1. 12. An alcohol of formula III Y—CO—X—CO—Arg(OH)—R  III inwhich Arg is L-arginyl; R is 2-benzothiazolyl; X—CO— is L-prolyl or(S)-azetidin-2-carbonyl; and Y—CO— is a group of formula IIa, IIb, IIcor IId;

and Arg(OH) indicates that the carbonyl portion of the L-arginyl groupis replaced by a hydroxymethylene group.
 13. A method of inhibitingthrombosis in a mammal comprising administering to a mammal in need oftreatment, an antithrombotic dose of a compound of formula I as claimedin claim 1.